Protein function is dependent on its three-dimensional structure. When a protein is synthesized in a mammalian cell, it first appears essentially as a linear polypeptide chain. The immature chain then folds under appropriate cellular conditions (pH, ionic strength, etc.). Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quaternary structures. Sometimes the protein folding occurs with the help of protein folding catalysts called molecular chaperones, which are proteins themselves. Out of thousands of possible three dimensional shapes, an average mature protein assumes only one conformation, which is often referred to as the native structure of the protein. This conformation of the protein molecule is rather fragile. Any alteration in the protein's native structure may lead to loss of the protein's biological activity, a phenomenon called denaturation. Since the native structure is maintained mostly by weak forces (hydrogen bonding, electrostatic and hydrophobic interactions), proteins can easily be denatured by small changes in their environment. Thus protein denaturation occurs in their purification, storage, use, and transport. A given protein sample may therefore contain appreciable amounts of denatured, inactive protein besides the active, functional form.
Extensive unfolding sometimes causes precipitation of the protein from solution. Denaturation is defined as a major change from the original native state without alteration of the molecule's primary structure, i.e., without cleavage of any of the primary chemical bonds that link one amino acid to another. Treatment of proteins with strong acids or bases, high concentrations of inorganic salts or organic solvents (e.g., alcohol, chloroform, or guanidine hydrochloride), heat, mechanical shearing, or irradiation, all produce denaturation to a variable degree. Loss of three-dimensional structure usually produces a loss of biological activity. A denatured enzyme is often without catalytic function.
With the growth of the biotechnology industry and the increased production of recombinant proteins, interest in the mechanisms by which a protein adopts its native structure has increased dramatically. A number of therapeutic proteins are currently being produced by recombinant DNA technology, by incorporating a copy of the human gene encoding a particular protein into a rapidly dividing host cell such as a bacterium. The genes are then transcribed into mRNA and translated into protein by the host cell.
Recombinant proteins overexpressed in Escherichia coli are often accumulated as insoluble particles called inclusion bodies. Since proteins in inclusion bodies are usually inactive, they must be solubilized by a denaturing agent and refolded to recover their native steric structures having biological activities. In bioprocesses it is important to obtain a high refolding efficiency and high throughput at high protein concentrations.
Various methods for renaturing denatured proteins in solution have been disclosed. Renaturation of the denatured proteins is accomplished with varying success, and occasionally with a return of biological function, by exposing the denatured protein to a solution that approximates normal physiological conditions. Renaturation of proteins using cyclodextrins in a detergent-free liquid medium has been described in U.S. Pat. No. 5,728,804. A high pressure-based method for the refolding of denatured proteins in solution was provided in U.S. Pat. No. 6,489,450.
Renaturation (refolding) processes can involve dispersing the protein inclusion bodies in a buffer in the presence of “refolding aids,” which can interact with the protein to enhance its renaturation. J. L. Cleland et al., Biotechnology, 10, 1013 (1992), reported that polyethylene glycol enhances refolding yields. Various sugars and detergents have also been employed in refolding. G. Zardeneta et al., J. Biol. Chem., 267, 5811 (1992); L. H. Nguyen et al., Protein Expression Purif., 4, 425 (1993). Recently, D. Rozema et al., J. Amer. Chem. Soc., 117, 2373 (1995), reported that sequential complexation of denatured carbonic anhydrase B with a quaternary amine detergent, CTAB, followed by addition of beta-cyclodextrin to the complex, caused reactivation of the enzyme. None of these methods have been used to refold proteins inside cells, either in vitro or in vivo.
A continuing need exists for methods and compositions for renaturation of denatured proteins. It is particularly important to discover non-toxic compounds and methods that aid renaturation of proteins in an aqueous solution. Such protein folding aids and methods should be inexpensive, non-toxic, and easily administered to the denatured protein sample.